Em muitos mamíferos, é notado um influxo de linfócitos no início da gestação, que é acompanhado por um processo de proliferação, diferenciação e migração destas células. Em camundongos estes linfócitos se distribuírem na região mesometrial de cada sítio de implantação (SIE). Estudos imunocitoquímicos indicaram que estes linfócitos são células Natural Killer, porém, sua morfologia e comportamento peculiar sugerem que elas representam uma subpopulação de NK específica do útero durante a gestação, sendo portanto denominadas células Natural Killer uterinas (uNK). A função conhecida para estas células em camundongos é a liberação de citocinas como IFN- que leva à dilatação das artérias espiraladas deciduais e manutenção da integridade decidual, contribuindo, portanto, para o desenvolvimento normal da decídua e da placenta. A infecção intra-uterina foi reconhecida como a causa primária de partos prematuros e corrobora para o desenvolvimento de leucomalácia periventricular e displasia broncopulmonar. Estudo recente em camundongos prenhes demonstrou que a injeção intra-uterina de LPS de E.coli provoca aumento da expressão de Fosfodiesterase 4B (PDE4B) e da atividade fosfodiestrase na interface materno-fetal, aumento nas concentrações de TNF-α, IL-1β, IL-6 e IL-10 no líquido amniótico o que induz o parto prematuro e a diminuição da viabilidade fetal. É esperado que a administração de LPS promova o aumento sistêmico e central de citocinas próinflamatórias, e que o animal responda com alterações endócrinas, imunológicas e comportamentais com o fito de favorecer o restabelecimento da homeostasia. Nesse contexto, o presente trabalho buscou avaliar os efeitos comportamentais da administração de LPS em camundongos, bem como os efeitos no útero prenhe e nas células uNK desses animais por meio do estudo morfológico, citoquímico e estereológico. O estabelecimento do comportamento febril foi identificado a partir da segunda hora após a administração de LPS, e esse evento ocorreu concomitante ao estabelecimento do comportamento análogo à ansiedade. Foi possível identificar nos animais tratados o comportamento de apatia e redução na capacidade exploratória, que somados ao comportamento depressivo, estabelecido após 1 hora da aplicação do LPS, fazem parte das alterações que constituem o comportamento doentio. O tratamento provocou também alterações no ambiente uterino, de tal forma que foi possível identificar um significativo aumento de células uNK imaturas na região do miométrio dos SIE. Além disso, o tratamento levou à diminuição da quantidade de células uNK maduras e senescentes nas três regiões estudadas dos SIE concomitante ao aumento de células uNK que possuíam padrão alterado na expressão de N-actilgalactosamina nos seus grânulos e superfície. O período onde foram encontradas tais alterações acompanhou o período onde houve perda na marcação de perforina, sugerindo a ocorrência de degranulação. A imunocitoquímica demonstrou que o tratamento com LPS parece ter induzido a despolimerização dos filamentos contrateis de α-actina causando relaxamento nos vasos que irrigam o ambiente uterino. / In mammalian, there is a lymphocyte influx in early pregnancy, which is accompanied by proliferation, differentiation and migration of these cells. These lynphocites distribute in mesometrial region of each embryo implantation site (EIS). Immunocytochemical studies indicated that these cells are Natural Killer cells, but their peculiar morphology and behavior suggest that they represent a uterus specific NK subpopulation during pregnancy called uterine Natural Killer cells (uNK). The known function for these cells in mice is cytokines releasing such as IFN- leading to decidual spiral arteries dilation and maintaining decidual integrity, contributing to normal development of decidua and placenta. Intrauterine infection was recognized as primary cause of preterm labor and supports the periventricular leukomalacia and bronchopulmonary dysplasia development. Recent study in pregnant mice showed that E.coli LPS intra-uterine injection causes increased expression of Phosphodiesterase 4B (PDE4B) and fosfodiestrase activity in maternal-fetal interface, increased TNF-α, IL-1β, IL-6 and IL-10 concentrations in amniotic fluid which leads to premature delivery and reduced fetal viability. It is expected that LPS administration promotes systemic and central proinflammatory cytokines increasing, and that the animal responds with endocrine, immune and behavioral disorders with the aim of promoting homeostasis restoration. In this context, the present study evaluated the behavioral effects of LPS administration in mice and the effects on pregnant uterus and uNK cells of these animals through morphological, cytochemical and stereological study. The feverish behavior establishment was identified from second hour after LPS administration, and this event was concomitant with anxiety-like behavior establishment. It was possible to identify treated animals apathy behavior, reduction in exploratory capacity and depressive behavior drawn 1 hour after LPS application as part of sickness behavior. The treatment also caused changes in the intrauterine environment, so that we could identify a significant increase of immature uNK cells in myometrium region of EIS. Furthermore, the treatment decreased the amount of mature and senescent uNK cells in the three EIS studied regions, concomitantly with the increasing granules and surface N-actilegalactosamine expression altered uNK cells. These changes were accompanied by the perforin labeling loss and a reduction on blood vessel α-actin labeling, suggesting respectively the uNK cell citotoxicity (perforin releasing) and actin contractile filament despolymerization causing relaxation of blood vessels that supplies the decidua and embryo. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

Type 2 (Th2) immune responses are required for immune defence against helminths, but can also have pathogenic effects in allergic conditions. This thesis examined two factors which may influence Th2 immunity at a cellular and molecular level: cross-talk between Natural Killer (NK) cells and dendritic cells (DCs) and the cell surface organisation of DCs. Cross-talk between NK cells and DCs is well-established to impact Th1 responses against tumours and infection; however the influence of this interaction during Th2 inflammation is unknown. To investigate this, human monocyte-derived DCs were stimulated in vitro with different pathogen-associated molecules; LPS or Poly(I:C) which polarise a Th1 response, or soluble egg antigen (SEA) from the helminth worm Schistosoma mansoni, a potent Th2-inducing antigen. These cells were then combined with autologous NK cells. Confocal microscopy showed polarisation of the NK cell microtubule organising centre (MTOC) and accumulation of LFA-1 at contacts between NK cells and immature or Th2-polarising DCs, but not Th1-polarising DCs, indicative of the assembly of an activating immune synapse. NK cells did not lyse DCs treated with LPS or Poly(I:C), but degranulated to and lysed both immature DCs and Th2 polarising DCs. Antibody blockade of NK cell activating receptors NKp30 and DNAM-1 prevented this lysis. Furthermore, depletion of NK cells in mice which were then transferred with Th2 polarising DCs led to an enhanced Th2 recall response. Thus, these data indicate a previously unrecognised role of NK cell cytotoxicity in restricting the pool of DCs involved in Th2 immune responses. Secondly, this thesis investigated the nanoscale organisation of MHC-II on the surface of Th1 and Th2 polarising DCs using ground state depletion super-resolution microscopy. MHC-II was relatively homogenously distributed across the membrane with no significant changes in clustering between immature, Th1 and Th2 polarising DCs. In contrast, imaging CD74, which can mediate internalisation of MHC-II, revealed increased expression and a more homogenous distribution of this receptor on the surface of Th2-polarising DCs compared to Th1-polarising DCs. These data suggest that changes in the clustering of CD74 could modulate MHC-II surface expression during Th2 responses. Overall, the results in this thesis indicate that both molecular and cellular level modulation of DC function contribute to the development of Th2 responses.

The purpose of this study was to investigate natural killer cell activity and the possible role of beta- endorphin in a natural model of autoimmune orchitis, the dark mink. An assay was developed to study natural killer cell activity in the mink and base-line levels of activity in this specie were determined. Natural killer cell activity was assessed in fertile mutation mink, primary infertile dark mink and secondary infertile Utah dark mink with autoimmune orchitis. The study included three sampling times: November, March and April. Natural killer cell activity was significantly lower in mink studied in April than it was in. mink studied in March or November, and there was a significant correlation of activity to fertility. The addition of beta-endorphin to natural killer cell cultures had no effect on activity. A preliminary study was done to determine concentration levels of beta-endorphin in mink plasma, pituitary, hypothalamus and testes. Measurable amounts were found in all tissue types studied. Although the number of samples was too limited to draw significant conclusions, there appears to be a trend toward lower beta-endorphin concentration, in pituitary tissue and plasma samples, of mink with autoimmune orchitis. Beta-endorphin levels did not appear to correlate with natural killer cell activity in the mink. Two assays, the natural killer assay and the blasto-genesis assay, were used to insure that the beta-endorphin preparation used in this study were active. Natural killer cell activity had been reported to be enhanced by the addition of beta-endorphin. In the present study natural killer cell activity of human peripheral blood mononuclear cells (PBMC) was enhanced in the presence of betaendorphin, suggesting that the peptide was active. In addition, the blastogenic response of human PBMC was enhanced by the addition of beta-endorphin to cultures in the present study.

Surgery precipitates a hypercoagulable state and has been shown to increase the development of cancer metastases in animal models, however mechanism(s) responsible for this are largely unknown. We hypothesize that the prometastatic effect of surgery may be secondary to postoperative hypercoagulable state. Surgical stress was induced in mice by partial hepatectomy or nephrectomy, preceded by intravenous injection of CT26-LacZ or B16F10-LacZ cells to establish pulmonary metastases with or without perioperative anticoagulation and their lung tumor cell emboli (TCE) were quantified. Fibrinogen and platelets were fluorescently labeled prior to surgical stress to evaluate TCE-associated fibrin and platelet clots. Surgery significantly increased metastases while anticoagulation with five different agents attenuated this effect. Fibrin and platelet clots were associated with TCE significantly more frequently in surgically stressed mice. Surgery promotes the formation of fibrin and platelet clots around TCE and this appears to be the mechanism for the increase in metastases seen following surgery.

Natural killer (NK) cells are lymphocytes of the innate immune system that play a major role in the destruction of both tumours and virally-infected cells. The cytotoxicity of NK cells is tightly controlled by signals received through activating and inhibitory receptors. NK cells express a variety of inhibitory receptors such as Ly49 receptors. Ly49 receptors bind to class I MHC molecules that expressed on normal cells. Using Ly49-deficient (NKCKD) mice we show that Ly49-KD NK cells successfully recognize and kill influenza virus-infected cells and that NKCKD mice exhibit better survival than wild-type mice. Moreover, influenza virus infection has a propensity to upregulate cell surface expression of MHC-I on murine lung epithelial cells in vivo. Significantly, we demonstrate increased lung damage of WT-mice versus NKCKD mice after influenza virus infection as determined by histological analyses. This data indicated that absence of Ly49 inhibitory NK receptors greatly enhances survival of infected mice.

Surgery precipitates a hypercoagulable state and has been shown to increase the development of cancer metastases in animal models, however mechanism(s) responsible for this are largely unknown. We hypothesize that the prometastatic effect of surgery may be secondary to postoperative hypercoagulable state. Surgical stress was induced in mice by partial hepatectomy or nephrectomy, preceded by intravenous injection of CT26-LacZ or B16F10-LacZ cells to establish pulmonary metastases with or without perioperative anticoagulation and their lung tumor cell emboli (TCE) were quantified. Fibrinogen and platelets were fluorescently labeled prior to surgical stress to evaluate TCE-associated fibrin and platelet clots. Surgery significantly increased metastases while anticoagulation with five different agents attenuated this effect. Fibrin and platelet clots were associated with TCE significantly more frequently in surgically stressed mice. Surgery promotes the formation of fibrin and platelet clots around TCE and this appears to be the mechanism for the increase in metastases seen following surgery.

Τόσο τα βλαστικά όσο και τα προγονικά κύτταρα συντονίζουν τον πολλαπλασιασμό και τη διαφοροποίηση για να δώσουν γένεση στον κατάλληλο αριθμό λειτουργικά εξειδικευμένων κυττάρων κατά την οργανογένεση. Διαφορετικές πειραματικές προσεγγίσεις ανέδειξαν τον ρόλο της πρωτεΐνης Geminin στη διατήρηση των προγονικών κυττάρων, τη συμμετοχή της στις αποφάσεις καθορισμού της αναπτυξιακής τους τύχης αλλά και την συμβολή της στην οργανογένεση. Αν και ο ακριβής μηχανισμός δεν είναι ξεκάθαρος, η Geminin φαίνεται να επηρεάζει την κατεύθυνση του πολλαπλασιασμού έναντι της διαφοροποίησης. Για να διαλευκανθεί ο ρόλος της Geminin in vivo στην κυτταρική διαίρεση και διαφοροποίηση των προγονικών κυττάρων, δημιουργήθηκαν ποντίκια που στερούνται την έκφραση της Geminin ειδικά στα κύτταρα της λεμφοειδούς σειράς μέσω Cre ανασυνδυασμού. Προηγούμενες μελέτες έδειξαν ότι η απουσία της Geminin έχει ελάχιστες επιπτώσεις στην ανάπτυξη και διαφοροποίηση των θυμοκυττάρων ενώ τα ώριμα περιφερειακά Τ κύτταρα που δεν εκφράζουν Geminin εμφανίζουν πολλές επιπτώσεις στον πολλαπλασιασμό μετά από in vitro ενεργοποίηση. Τα φυσικά φονικά κύτταρα (Natural Killer Cells) αναπτύσσονται στο μυελό των οστών από κοινά αρχέγονα κύτταρα και καθορίζονται από την ικανότητά τους να σκοτώνουν καρκινικούς στόχους χωρίς προηγούμενη ενεργοποίηση. Τα κύτταρα αυτά παίζουν σημαντικό ρόλο στις μη ειδικές αποκρίσεις και πέρα από την ισχυρή κυτταροτοξική δράση έναντι ευαίσθητων κυττάρων-στόχων χαρακτηρίζονται από την ικανότητα να απελευθερώνουν διάφορες κυτταροκίνες αποκρινόμενα άμεσα σε ενδοκυτταρικές μολύνσεις. Επιπρόσθετα, αποτελούν έναν κρίσιμο κυτταρικό πληθυσμό για την προστασία των ιικών μολύνσεων, καθώς αναγνωρίζουν με ειδικό τρόπο ιικά μολυσμένα κύτταρα και τα σκοτώνουν απευθείας. Για τους λόγους αυτούς παρουσιάζει ενδιαφέρον ο έλεγχος των τυχόν επιπτώσεων της απουσίας της Geminin στα ΝΚ κύτταρα. Με βασικό πειραματικό εργαλείο την κυτταρομετρία ροής, μελετήσαμε την αναπτυξιακή πορεία των φυσικών φονικών κυττάρων απουσία της Geminin. Σε κυτταρικά εναιωρήματα από σπλήνα και μυελό των οστών από Fl/koCD2Cre και Fl/WT πειραματόζωα, NK1.1+TCRβ- ΝΚ κύτταρα αναλύονται με κυτταρομετρία ροής για τον έλεγχο της έκφρασης μορίων-δεικτών των ώριμων ΝΚ κυττάρων. / The interplay of proliferation and differentiation is essential for normal development and organogenesis. Stem cells as wells as progenitor cells coordinate these cellular decisions to give rise to the appropriate number of differentiated cells with specific function. Geminin was identified through two parallel lines of investigation as both a regulator of DNA replication and a regulator of differentiation of neural cells. In order to address the in vivo role of Geminin in regulating progenitor cell self-renewal and differentiation decisions in different cellular systems, have been generated mice that allow the conditional inactivation of the mouse Geminin gene in cells giving rise to the lymphoid lineage. Natural Killer Cells are lymphocytes of the innate immunity with effector function such as perforin-dependent cytotoxicity and interferon-γ secretion. Several lines of evidence implicate them in the early control of virus infection, in tumor immunoserveillance and in the regulation of immune responses. Mature NK cells express a wide variety of cell surface receptors that enable them to recognize targets expressing low surface amounts of major histocompatibility complex class I molecules or high surface amounts of molecules induced by stress or microbial molecules. NK cells generation occurs in the bone marrow from hematopoietic stem cells. In this study, we shed light to the in vivo role of Geminin in the development and differentiation of Natural Killer Cells. Cell suspensions from bone marrow and spleen from Fl/koCD2Cre mice that lack Geminin expression in the lymphoid lineage and Fl/WT mice that serves as controls were analyzed with flow cytometry. NK1.1+TCRβ- ΝΚ cells were analysed for surface receptors, markers of mature NK cells.

In this thesis, the monocrotaline (MCT)-induced rat model of pulmonary arterial hypertension (PAH) was used to investigate the role of innate immunity in the pathogenesis of PAH and the mode of action of experimental therapies. The first section of this thesis is an investigation of the therapeutic mechanism of human, early and late-outgrowth endothelial progenitor cells (EPCs) in the MCT-induced, nude rat model of PAH. While late-outgrowth EPCs provided no therapeutic benefit in this model, early EPCs (E-EPCs) prevented the elevation of right ventricular systolic pressure (RVSP, P<0.001) and right ventricular (RV) hypertrophy (P<0.01). Ablation of natural killer (NK) and natural killer T cells with anti-asialo GM-1 antiserum (ASGM-1) enhanced human cell retention in the lung but abrogated the therapeutic capacity of E-EPCs. In vitro studies demonstrated that E-EPCs are similar to monocyte-derived regulatory dendritic cells (DCs) and possess the capacity to stimulate both autologous and rat NK cells in co-culture. Imatinib mesylate has been reported to reverse established PAH both clinically and in the MCT model. Imatinib can also induce NK activation through inhibition of c-kit signaling in DCs, suggesting that imatinib and the DC-like E-EPCs may prevent PAH through a similar, NK-mediated mechanism. In the second section of this thesis, imatinib prevented MCT-induced increases in RVSP (P<0.001) and RV hypertrophy (P<0.01) in immunocompetent Fisher 344 rats, but not in nude rats or Fisher rats following ablation of NK cells and T lymphocytes with ASGM-1. These data suggest that the stimulation of NK activity by imatinib is insufficient to prevent disease in the absence of T lymphocytes. Hyaluronan (HA) fragments are a potent inflammatory stimulus, capable of inducing macrophage activation and DC maturation. In the third section of this thesis, HA synthesis and degradation were investigated in the MCT model of PAH. While the early stages of disease were characterized by enhanced hyaluronidase-1 activity and a loss of high molecular weight (HMW) HA, severe disease was associated with HMW HA synthesis and HA accumulation in the lungs. The early degradation of HMW HA may drive inflammation and stimulate pathological vascular remodeling in PAH.

In this thesis, the monocrotaline (MCT)-induced rat model of pulmonary arterial hypertension (PAH) was used to investigate the role of innate immunity in the pathogenesis of PAH and the mode of action of experimental therapies. The first section of this thesis is an investigation of the therapeutic mechanism of human, early and late-outgrowth endothelial progenitor cells (EPCs) in the MCT-induced, nude rat model of PAH. While late-outgrowth EPCs provided no therapeutic benefit in this model, early EPCs (E-EPCs) prevented the elevation of right ventricular systolic pressure (RVSP, P<0.001) and right ventricular (RV) hypertrophy (P<0.01). Ablation of natural killer (NK) and natural killer T cells with anti-asialo GM-1 antiserum (ASGM-1) enhanced human cell retention in the lung but abrogated the therapeutic capacity of E-EPCs. In vitro studies demonstrated that E-EPCs are similar to monocyte-derived regulatory dendritic cells (DCs) and possess the capacity to stimulate both autologous and rat NK cells in co-culture. Imatinib mesylate has been reported to reverse established PAH both clinically and in the MCT model. Imatinib can also induce NK activation through inhibition of c-kit signaling in DCs, suggesting that imatinib and the DC-like E-EPCs may prevent PAH through a similar, NK-mediated mechanism. In the second section of this thesis, imatinib prevented MCT-induced increases in RVSP (P<0.001) and RV hypertrophy (P<0.01) in immunocompetent Fisher 344 rats, but not in nude rats or Fisher rats following ablation of NK cells and T lymphocytes with ASGM-1. These data suggest that the stimulation of NK activity by imatinib is insufficient to prevent disease in the absence of T lymphocytes. Hyaluronan (HA) fragments are a potent inflammatory stimulus, capable of inducing macrophage activation and DC maturation. In the third section of this thesis, HA synthesis and degradation were investigated in the MCT model of PAH. While the early stages of disease were characterized by enhanced hyaluronidase-1 activity and a loss of high molecular weight (HMW) HA, severe disease was associated with HMW HA synthesis and HA accumulation in the lungs. The early degradation of HMW HA may drive inflammation and stimulate pathological vascular remodeling in PAH.

No description available.